Among the "allosteric effectors" regulating Hemoglobin oxygen affinity, chloride ions have long been known to have a significant physiological role. From in vitro studies of the relation between O2 binding and C1- activity, it has been thought that some 1.6 Chlorides bind to stabilize the "deoxy" or "T" form [1-4]. Last year we discovered that the oxygen affinity of Hemoglobin correlates with eater activity in the presence of several neutral solutes. We inferred a change in protein hydration in the deoxy T state to fully oxygenated R state transition of some 60 - 65 waters. We were forced to ask, then, whether salt could have an osmotic side-effect similar to that seen with neutral solutes. We therefore re- examined the regulatory action of chloride, explicitly including changes in protein hydration and the dependence of water activity on added salt. We now find that an alternate description of the data has only a single allosteric chloride ion (delta c1 = -1.22 + 0.02 C1-) directly linked with oxygenation during the deoxy-to-oxy transition, while some 65.2 + 2.4 additional water molecules bind to the protein [ (5)]. Within this analysis, the C1- -regulated loading of four oxygens can be described by the reaction, Hb.C1 + 4O2 + 65H2O <==> Hb.4O2.65H2O + C1-. Far more important than simply a matter of Hemoglobin or chloride alone, we must now face the possibility of a general bias in gauging the action of "effectors" of protein function, a bias from neglect of solvation and the new hydration forces that seem to be ubiquitous among bio-molecules.